Removal of nucleic acid contaminants

ABSTRACT

A method of analysing a nucleic acid sample obtained from a site comprising the step of pretreating the sample to remove or inactivate contaminating nucleic acids originating from the site. The pre-treatment may comprise chemical, enzyme or a physical treatment that acts to selectively or preferentially remove or inactivate the contaminating nucleic acids.

FIELD OF THE INVENTION

The present invention relates to improved methods for collecting andanalysing nucleic acid samples such as nucleic acid samples of forensicvalue from crime scenes. The present invention also relates to databasescontaining data obtained using the improved methods.

BACKGROUND OF THE INVENTION

The analysis of DNA and in particular DNA identification such as DNAprofiling, DNA fingerprinting and genetic profiling (hereinafterreferred to as “DNA profiling”), is used in many areas of research andcommercial activity including agriculture, veterinary science, medicineand forensics. In agriculture and veterinary science, DNA profiling isused to identify plant and animal genotypes for breeding andidentification purposes and in medical science DNA profiling is used forvarious purposes including identification of related individuals.

DNA profiling has also become an important tool in forensic science andlaw enforcement. In DNA forensics, DNA isolated from crime scenes can beamplified and visualised using techniques such as polymerase chainreaction (“PCR”) and gel electrophoresis and the resulting profile or“fingerprint” can be used to place a suspect at a crime scene.

The methods used to analyse DNA, such as PCR amplification, are simple,quick and highly sensitive so they can be carried out using smallsamples or samples that have been partially degraded. However, thesensitivity of DNA analysis techniques also has potential disadvantages.For example, strict contamination control is essential when undertakinganalysis using PCR as contaminants in the starting sample will also beamplified. This is particularly so when the contaminants are ampliconsderived from PCR as these are amplified with high efficiency during PCR.Consequently, forensic and other testing laboratories go to considerableeffort to prevent the contamination of samples during both thecollection and processing stages.

When samples are contaminated and the source of the contamination can bereadily identified (e.g. the person collecting the sample or running thePCR) samples can be taken from those individuals and the PCR bandsgenerated by that individual subtracted from the test results to correctfor the contamination. In a crime scene where multiple individuals maybe present, most contaminating individuals (samples not belonging to theperpetrator/s) can be identified and the effects of that contaminationeliminated from further analysis.

However, to date it has not been recognised that DNA samples could bepurposefully contaminated with the intention of confounding future DNAanalysis. For example, microsatellite PCR amplicons generated by the useof a commercially available kit on a standard DNA control or randomtissue sample (hereafter referred to as “perfect amplicons”) could beadded to water or another solvent and used to contaminate a sample orthe area from which a sample for future DNA analysis is to be taken. Inthe example of a contaminated crime scene, these perfect amplicons wouldbe collected along with the forensic sample during the collection ofsamples for forensic analysis from the scene. These perfect ampliconswould be efficiently isolated using current DNA extraction methodswidely used in DNA forensics and may be present in a vast excess overthe DNA of true forensic value from the crime scene. Furthermore, beingshort perfect amplicons, they would be amplified with greater efficiencythan any genomic DNA present. Upon amplification the resulting profilewould consist almost entirely of the contaminating perfect amplicon DNA.Depending on the nature of the contaminating amplicons and theirconcentration in the sample(s), the resultant profile may beindistinguishable from a real profile, or may render the identificationof the genuine profile of the forensic sample difficult or impossible todetermine.

Although current forensic testing usually uses PCR amplification ofselected microsatellite regions from the forensic sample nucleic acid,other methods in use or development such as mitochondrial DNAsequencing, single nucleotide polymorphism analysis, low copy number PCRand other methods known to those familiar with DNA analysis methods arealso susceptible to this form of contamination.

When not accounted for in the testing process, the potential forcontamination of this nature compromises the validity of the DNAanalysis and substantially limits the strength of any conclusions drawntherefrom. For example, crime scenes could be contaminated with nucleicacid with a view to confounding future forensic analysis and limitingthe legal value of the DNA analysis results used in court proceedings.

At present, the techniques used to analyse nucleic acid samples forforensic purposes do not reliably distinguish between nucleic acidsadded to contaminate the sample and the true target nucleic acids in asample. Laboratory procedures currently used are designed to minimisecontamination of samples in the laboratory and will not be effective inremoving contamination when contaminated samples are presented to thelaboratory for analysis.

The most common and widely used system to reduce or remove contaminationwith PCR derived amplicons relies on the incorporation of DNA nucleotideanalogues such as deoxy-uracil triphosphate (dUTP) into DNA during PCRamplification. However, this method is also designed to preventlaboratory cross contamination and does not address the problemsencountered when a sample has been contaminated with nucleic acidcontaining deoxy-thymine triphosphate (thymine, dTTP) instead of dUTP.Consequently this method of contamination control is easily avoided andis not effective in removing contamination when dTTP containing samplesare presented to the laboratory for analysis.

Thus, the present invention seeks to provide methods that deal with thepreviously unrecognised problem of reliably detecting the presence ofcontamination and processing nucleic acid samples that have thepotential of being, or have been, purposefully contaminated to removethe contaminant.

SUMMARY OF THE INVENTION

The present invention provides a method of analysing a nucleic acidsample obtained from a site comprising the step of pretreating thesample to remove or inactivate contaminating nucleic acids originatingfrom the site.

The present invention also provides a method of screening a nucleic acidsample for contaminants that have been purposefully introduced into thesample, the method comprising the step of treating the sample to detectthe contaminants.

The methods of the present invention may be broadly applied and inparticular may be applied to forensics and animal, plant and humannucleic acid testing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a gel electrophoresis of various PCR amplifications ofuncontaminated and contaminated samples;

FIG. 2 depicts a gel electrophoresis of another series of PCRamplifications of treated and untreated contaminated samples;

FIG. 3 depicts a gel electrophoresis of various PCR amplifications ofuncontaminated and contaminated samples

FIG. 4 depicts a gel electrophoresis of various PCR amplifications ofuncontaminated and contaminated samples;

FIG. 5 depicts a gel electrophoresis of various PCR amplifications ofuncontaminated and contaminated samples;

FIG. 6 depicts a gel electrophoresis of various PCR amplifications ofcontaminated samples that have been treated according to one embodimentof the invention; and

FIG. 7 depicts a gel electrophoresis of various PCR amplifications ofcontaminated samples that have been treated according to one embodimentof the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of analysing a nucleic acidsample obtained from a site comprising the step of pretreating thesample to remove or inactivate contaminating nucleic acids originatingfrom the site.

For the purposes of the present invention the phrase “contaminatingnucleic acid/s” is defined as nucleic acid that has been introduced to asite or a sample to confound future analysis of target nucleic acidspresent at the site or in the sample. The contaminating nucleic acid maybe cell bound, free or substantially free from other cell components andmay be deoxyribonucleic acid (DNA), ribonucleic acid (RNA), proteinnucleic acid (PNA), locked nucleic acid (LNA) or any other nucleic acidcontaining composition such as those containing natural nucleotides(e.g. dATP, dCTP, dTTP, dUTP, dGTP) or nucleotide/nucleoside analoguesthat are capable of detection during testing procedures.

When the contaminating nucleic acid is free or substantially free fromother cell components it may be in a form that is particularly welladapted for amplification via PCR or some other amplification processthat is used in forensic analysis. One particular example of this typeof contaminating nucleic acid is an amplicon derived from a PCR oranother DNA amplification process and in particular a degradationresistant amplicon that has been specifically designed to persist at asite or in a sample. Synthetic DNA, RNA or PNA may also be used.

The contamination addressed by the present invention may confound anynucleic acid analysis protocol where samples may be contaminated. Thus,while specific mention is made herein of PCR, it will be appreciatedthat the same contamination could be used to alter the results of otheranalysis methods such as, but not limited to, mitochondrial DNAsequencing, single nucleotide polymorphism (SNP) analysis and low copynumber PCR.

When the contaminating nucleic acid is cell bound it may also be in aform that is particularly well adapted for amplification via PCR or someother amplification process that is used for analysing nucleic acids.One particular example of this type of contaminating nucleic acid is abacterial preparation where the bacteria have been engineered to containone or more multicopy plasmids each comprising one or more ampliconsable to be amplified during standard forensic PCR processes.

The pre-treatment may be varied depending on the nature of thecontaminating nucleic acids that require removal or inactivation. Thus,when the contaminating nucleic acids are free or substantially free fromother cell components, the pre-treatment may comprise treating thesample to preferentially remove or inactivate nucleic acids that arefree or substantially free from other cell components. Such treatmentsmay be one or more treatments selected from the group comprising: (i)enzymic treatments such as contacting the sample with enzymes thatpreferentially breakdown free nucleic acids e.g. DNAses, RNAses,exonucleases and endonucleases; (ii) physical treatments that removefree contaminating nucleic acid from the sample based on differencesbetween physical characteristics of the contaminating nucleic acid andthe target nucleic acid such as charge, density, weight and size and theactual techniques used may be selected from the group comprisingcentrifugation (e.g. with centricon 100 columns), washing, filtrationand chromatography such a gel filtration chromatography; or (iii)chemical treatments such as the use of sodium hydroxide, sodiumhypochlorite, sodium metabisulfite, sodium bisulfite or ammoniummetabisulphite, detergents (e.g. Tween 20, Alcanox or SDS) as well asproprietary products designed to remove nucleic acids form surfaces suchas DNA Zap, RNA Zap, DNA Free or RNA Free (Ambion Inc., Austin, Tex.,USA).

When the contaminating nucleic acids are free from other cell componentsthen the pre-treatment may comprise contacting the sample with nucleicacid probes that preferentially bind to the contaminating nucleic acidsand render them removable from the sample. This is particularlyappropriate for the removal of contaminating nucleic acids in the formof PCR derived amplicons.

Thus, the present invention also provides a method of analysing anucleic acid sample obtained from a site comprising the step ofcontacting the sample with a nucleic acid probe that preferentiallybinds to the contaminating nucleic acids and renders them removable fromthe sample.

The choice of probe is entirely dependent on the nature of thecontaminant. However, it is envisaged that the most common contaminantswill be derived from the commercially available forensic DNA test kitsand in particular the positive controls that can be readily amplifiedvia PCR. Thus, the probes may be designed to specifically hybridise tothe amplification product of the positive control from a proprietarykit. In the event that a new contaminant is produced then it would benecessary to first characterise the contaminant to enable appropriateprobes to be designed for use in the method.

The nucleic acid probe may be labelled to aid in its removal from thesample. Suitable labels include biotin/streptavidin.

In one particular form of the invention the contaminating nucleic acidbound to the labelled probe is removed through the use of achromatography column adapted to specifically bind the label.

When the contaminating nucleic acids are cell bound or otherwise cellassociated in a way that prevents or hampers their removal orinactivation, such as if contained in bacterial cells, then additionalpre-treatments may be required. For example, when the contaminatingnucleic acids are contained within bacterial cells, an additional stepto selectively lyse the bacterial cells may be employed. Once thebacterial cells have been lysed the techniques discussed above could beused to complete the pre-treatment. Contaminants in the form ofbacterial cells may also be removed by using a filter that selectivelyremoves the bacterial cells from the sample.

Once the sample has been pre-treated it can be treated according tostandard techniques for nucleic acid analysis. Thus, the presentinvention also provides a method of analysing a nucleic acid sampleobtained from a site comprising the steps of:

-   -   (i) pre-treating the sample to remove or inactivate        contaminating nucleic acids originating from the site; and    -   (ii) characterising the target nucleic acids in the sample.

The nucleic acids in the sample can be characterised by any one of arange of techniques that are presently in use in the field. Thesetechniques generally involve isolating the target nucleic acid and thentreating it such that it can be conveniently characterised. Thesetechniques and procedures are well known by those skilled in the art.

The target nucleic acid may be isolated using standard extractionprotocols that involve lysing the cells to free the nucleic acid andthen separating the nucleic acid from other cellular material. Onceisolated, to increase the amount of target nucleic acid, the targetnucleic acid may be selectively amplified using PCR or some othertechnique that is able to replicate the target DNA to increase theamount available for further analysis. Once amplified the target nucleicacid can be visualised using gel electrophoresis. Proprietary DNAprofiling or fingerprinting kits can also be used to perform this partof the method.

Screening Methods

Rather than applying the method of the present invention to all samplestaken for nucleic acid analysis, it may be preferred to screen samplesfor contamination prior to nucleic acid analysis. By applying thismethod, samples that have been contaminated can be identified andhandled accordingly.

Thus, the present invention also provides a method of screening anucleic acid sample for contaminants that have been purposefullyintroduced into the sample, the method comprising the step of treatingthe sample to locate the contaminants.

Various treatments may be applied to a sample to screen for contaminantsincluding the use of a detectable probe designed to selectivelyhybridise to the contaminant. As indicated above, it is expected themost common contaminants will be sourced from commercial DNA analysiskits so the design of probes for this purpose will be routine to thoseskilled in the art. Alternatively, the wash solutions, filtrate,chromatography column eluate or other products resulting from theprocedures used to remove potential contaminants could be tested for thepresence of the contaminants.

Databases

The method of the present invention allows for the accurateidentification of nucleic acids and counters the effects of contaminantsthat may have been introduced into a sample with a view to confoundingtheir analysis. DNA fingerprint databases currently in existence includefingerprints that have been determined using methods that do not accountfor the potential problems of contamination. Given the possibility ofcontamination, the conclusions drawn from fingerprints in the currentdatabases may be queried. This could be a particular problem in courtproceedings where DNA fingerprint evidence has been used to identify aperpetrator. It is possible that DNA analysis performed with protocolsthat do not account for purposeful contamination may be heldinadmissible.

Thus, the present invention also provides a database comprising theresults of at least one analysis generated from a method according tothe present invention, such as DNA fingerprint.

Preferably, the database is computerised for ease of use and comprisesfingerprints of known perpetrators. However, the database can containany data obtained through the use of the method of the presentinvention.

Kits

The method of the present invention may be conveniently performed usinga kit comprising a series of reagents necessary to carry out the method.Thus, the present invention also provides a nucleic acid analysis kitcomprising a means to remove a nucleic acid contaminant from a sample tobe subjected to analysis.

The means may be varied and includes those discussed herein such aslabelled probe adapted to bind to the contaminant and thus aid in itsremoval. Alternatively, the means may comprise an enzyme or chemicalthat can be added to the sample and inactivate of remove the contaminantpreferentially or selectively relative to the target nucleic acid.

The method of the present invention is generally applicable to methodsfor identifying or analysing nucleic acid samples. Described hereunder,are particular applications that demonstrate the broad application ofthe present invention.

Forensics

The method of the present invention may be of particular use in theanalysis of target nucleic acid obtained from crime scenes. Thus, thepresent invention also provides a method of analysing a nucleic acidsample obtained from a site in the form of a crime scene comprising thestep of pretreating the sample to remove or inactivate contaminatingnucleic acids originating from the crime scene.

For the purposes of the present invention the phrase “crime scene” isdefined to include sites where a crime has been committed or other sitesaway from the crime scene, where nucleic acid of forensic value relevantto the crime may be found.

In addition to carrying out the method of analysis described above inrelation to samples taken from a crime scene it may also be useful whenassessing crime scenes to screen the crime scene for contaminatingnucleic acid. To date, as this potential problem has not been recognisedno specific screening takes place.

Thus, the present invention also provides a method of analysing a crimescene comprising the step of screening the crime scene for contaminatingnucleic acids.

The screening step may be carried out by any means apparent to thoseskilled in the art for detecting contaminating nucleic acids at a crimescene. One such technique involves taking a sample from a point in thecrime scene that would not normally contain nucleic acids. This form ofscreen is particularly appropriate for locating contaminating nucleicacids, such as amplicons, that have been sprayed in liquid form at acrime scene.

In such situations contamination of crime scenes by perfect ampliconmaterial can be detected by testing samples collected from areas withinthe crime scene surrounding areas in which target nucleic acids arelocated e.g. sections of walls or floors adjacent a blood stain. Thegeneration of significant DNA profiles from such areas that would not beexpected to contain high levels of DNA may indicate a contaminated crimescene.

In situations where individual tissue samples (eg blood, hair etc) havebeen contaminated by perfect amplicons, contamination may be detected byhybridisation with microsatellite probes or amplification with primersthat bind to the primer sequences of known fingerprinting kits, underconditions such that the cells in the tissue sample are not disruptedsufficiently to release significant amounts of genomic DNA (e.g. reducedtemperatures).

Veterinary

The method of the present invention may also be applied to the analysisof target nucleic acid obtained from animals. Thus, the presentinvention also provides a method of analysing a nucleic acid sampleobtained from a site in the form of an animal comprising the step ofpretreating the sample to remove or inactivate contaminating nucleicacids.

Nucleic acid samples from animals are analysed for a wide range ofpurposes. Animals of a particular species or breed may be assessed by apotential buyer or breeder to confirm their genotype. Furthermore,commercial herds or products therefrom such as meat may require analysisto assess if they qualify for a government sponsored subsidy or thatthey meet certain regulatory requirements, such as GMO's or quarantinestandards. In all of these situations there is a motive for a person totamper with the samples for monetary or some other gain.

Agriculture

The method of the present invention may also be applied to the analysisof target nucleic acid obtained from plants. Thus, the present inventionalso provides a method of analysing a nucleic acid sample obtained froma site in the form of a plant such as a seed comprising the step ofpretreating the sample to remove or inactivate contaminating nucleicacids.

Nucleic acid samples from plants are analysed for a wide range ofpurposes. Plants of a particular species or variety may be assessed by apotential buyer or breeder to confirm their genotype. Furthermore,commercial crops or products therefrom may require analysis to assess ifthey qualify for a government sponsored subsidy or that they meetcertain regulatory requirements, such as GMO's or quarantine standards.In all of these situations there is a motive for a person to tamper withthe samples for monetary or some other gain.

Parentage Testing

The method of the present invention may also be applied to the analysisof target nucleic acid obtained from humans for assessing parentage.Thus, the present invention also provides a method of analysing anucleic acid sample obtained from a site in the form of a humancomprising the step of pretreating the sample to remove or inactivatecontaminating nucleic acids.

There are obvious motives for a person to tamper with a sample taken forassessment of parentage. Whilst the mechanics of tampering with samplesfor this purpose may be slightly more complicated, the method of thepresent invention also solves the problems that could lead to the abuseof this form of nucleic acid testing.

The invention will now be described with reference to two examples. Thedescription of the examples is in no way limiting on the more generaldescription of the invention in the preceding paragraphs.

EXAMPLES Example 1 Demonstration of PCR Amplification of ContaminatingMicrosatellite PCR Products During Amplification of Genomic DNAMicrosatellite Loci

Materials and Methods:

Template genomic DNA was isolated from muscle tissue of feral cat GD450.This cat carries alleles 25/21 at the locus FCA 69H (GenBank AF130500)carried by the cat chromosome B4. Genomic DNA was extracted from 4.5 mgof muscle tissue using the MasterPure™ DNA Purification Kit (EpicentreTechnologies, Madison, Wis., US). The manufacturers recommended protocolfor tissue extraction was followed with the exceptions that digestion ofthe tissue sample with Proteinase K was carried out overnight at 65° C.and protein was pelleted by centrifugation at 4° C.

Three different contaminants were produced by PCR amplification ofselected microsatellite loci from genomic DNA isolated from tissue of 3feral cats:

-   -   Cat MV1 (alleles 25/11): allele 25 in common with GD450    -   Cat MV4 (alleles 31/21): allele 21 in common with GD450    -   Cat MV5 (alleles 27/11): no allele in common with GD450

The contaminant microsatellite PCR products were approximated to have afinal concentration of 50 ng/μl.

PCR conditions and primer sequences used in this study are given inMenotti-Raymond, M.; David, V. A.; Lyons, L. A.; Schaffer, A. A.;Tomlin, J. F.; Hutton, M. K.; O'Brien, S. J. (1999). A Genetic LinkageMap of Microsatellites in the Domestic Cat (Felis catus). Genomics 57(1), 9-23. Medline 99208656.

Reactions containing 1 μl of GD450 genomic DNA and various dilutions ofsingle or mixed contaminant microsatellite PCR products were prepared asdescribed in Table 1. TABLE 1 Gel Genomic Contaminant dilution lane DNAVol. Contaminant (template Vol.) Comments 1 1 ul None n/a GD450 Positivecontrol 2 1 ul MV1 10⁻¹⁵ (1 ul) GD450 only 3 1 ul MV1 10⁻¹² (1 ul) GD450only 4 1 ul MV1 10⁻⁹ (1 ul) GD450 only 5 1 ul MV1 10⁻⁶ (1 ul) GD450/MV1mixed profile 6 1 ul MV1 10⁻³ (1 ul) GD450/MV1 mixed profile 7 1 ul MV11 (1 ul) GD450/MV1 mixed profile 8 1 ul MV4 10⁻¹⁵ (1 ul) GD 450 only 9 1ul MV4 10⁻¹² (1 ul) GD450 only 10 1 ul MV4 10⁻⁹ (1 ul) GD 450 only 11 1ul MV4 10⁻⁶ (1 ul) GD450 only 12 1 ul MV4 10⁻³ (1 ul) GD450/MV4 mixedprofile 13 1 ul MV4 1 (1 ul) GD450/MV4 mixed profile 14 1 ul MV5 10⁻¹⁵(1 ul) GD450 only 15 1 ul MV5 10⁻¹² (1 ul) GD450 only 16 1 ul MV5 10⁻⁹(1 ul) GD450 only 17 1 ul MV5 10⁻⁶ (1 ul) GD450/MV5 mixed profile 18 1ul MV5 10⁻³ (1 ul) GD450/MV5 mixed profile 19 1 ul MV5 1 (1 ul)GD450/MV5 mixed profile 20 1 ul Lanes 20-25 10⁻¹⁵ (1 ul each)Amplification failure 21 1 ul Mix of: 10⁻¹² (1 ul each) Amplificationfailure 22 1 ul MV1 + MV4 + MV5 10⁻⁹ (1 ul each) Amplification failure23 1 ul 10⁻⁶ (1 ul each) Amplification failure 24 1 ul 10⁻³ (1 ul each)GD450/MV1/MV4/MV5 profile 25 1 ul 1 (1 ul each) Too much template 26none none n/a No DNA Negative control

Results

The results of PCR amplification of various mixtures of cat GD450genomic DNA and microsatellite PCR product contaminants (see Table 1)are shown in FIG. 1. Short alleles (few repeats) give a strongerfluorescent signal if the template is a PCR product possibly due toincreased efficiency of amplification.

All samples tested contained equivalent amounts of genomic DNA from catGD450 and various amounts of contaminant PCR product generated from oneor more cats MV1, MV4, MV5 (Table 1). PCR amplification of genomic catGD450 DNA alone generated the expected bands for alleles 25 and 21 (FIG.1, lane 1). In the presence of low concentrations of the contaminant(10⁻¹⁵-10⁻⁹) the GD450 profile was the only profile present. At higherconcentrations (10⁻⁶, 10⁻³, undiluted) both the GD450 and thecontaminant profile were present, making it difficult, or impossible, todetermine the correct GD450 profile.

In mixtures containing MV1 or MV5 microsatellite PCR products (Lanes2-7, and 14-19, respectively) the contaminant profile was evident whenundiluted PCR (Lanes 7 and 19, respectively) product was added as wellas dilutions of 10⁻³ and 10⁻⁶ (Lanes 5, 6, and 17, 18, respectively).When MV4 was the source of the contaminant it was detected in onlyundiluted and 10⁻³ samples (Lanes 12 and 13). When all threecontaminating PCR products were mixed (total of 3 μl of PCR productadded) the amount of contaminant appeared to inhibit the PCRamplification (Lane 25) but at reduced concentrations (Lane 24) theprofile consisted of a combination of the three individual profiles. Inlane 24, the alleles (11 and 25) present in more than one template(MV1+MV5 and MV1+GD450, respectively) give stronger signals than alleles31, 27, 21 present in only one template due to the presence of multipleproducts.

These results clearly demonstrate that contaminating microsatellite PCRproducts are efficiently amplified during subsequent amplification formicrosatellites from genomic DNA of cat GD450. In some of these mixturessubsequent PCR amplification resulted in a combination of the individualprofiles such that the correct profile of test genomic DNA from catGD450 is effectively masked. There is a clear relationship between theamount of contaminating microsatellite PCR product added to the genomicDNA and the amplification of the contaminant, with less contaminantresulting in reduced amplification of the contaminant in subsequent PCRamplifications. The amount of contaminant required to achieve thismasking effect is extremely small. The undiluted contaminant had aconcentration of approximately 50 ng/μl. At a dilution of 10⁻⁶ only 50femtograms of PCR product was present. Since a typical PCR reactionwould contain approximately 1 ng -100 ng of genomic DNA it is clear thatonly trace amounts of contaminating PCR product are required to mask thegenuine GD450 profile.

Example 2 Demonstration That Contaminating Microsatellite PCR Productsare Extracted With Genomic DNA and Efficiently Amplified DuringAmplification of Genomic DNA Microsatellite Loci

Materials and Methods

Template genomic DNA was isolated from muscle tissue of feral cat.Genomic DNA was isolated from cat GD450 that scores 25/21 at the locusFCA 69H (GenBank AF130500) carried by the cat chromosome B4 or fromferal cat MV5 which gives 27/11 at the same locus. The contaminant foruse in these experiments was produced by PCR amplification of selectedmicrosatellite loci from DNA of cat MV5 and diluted with water to give afinal concentration of 20 ng/μl.

PCR conditions and primer sequences used in this study are given inMenotti-Raymond, M.; David, V. A.; Lyons, L. A.; Schaffer, A. A.;Tomlin, J. F.; Hutton, M. K.; O'Brien, S. J. (1999). A Genetic LinkageMap of Microsatellites in the Domestic Cat (Felis catus). Genomics 57(1), 9-23. Medline 99208656.

Extraction of Genomic DNA and Contaminant Removal

The genomic DNA of the cat GD450 was extracted from 4.5 mg of muscletissue with the MasterPure™ DNA Purification Kit (EpicentreTechnologies, Madison, Wis., US) using the modified protocol describedin Experiment One. The Masterpure kit provided a more stringent DNAextraction method than the phenol or Chelex™ extraction methodsrecommended in forensic kits such as the AmpFISTR Profiler Plus™ Kitfrom Applied Biosystems and should result in less carry through of anycontamination during the genomic DNA extraction procedure.

Seven different reaction tubes were setup:

-   -   1. Tissue (cat GD450) alone to obtain non contaminated genomic        DNA profile    -   2. 5 ul (100 ng) of cat MV5 contaminant DNA alone    -   3. Tissue (cat GD450)+5 ul (100 ng) of MV5 contaminant    -   4. Tissue (cat GD450)+5 ul (100 ng) of MV5 contaminant digested        with Hae III (Promega)    -   5. Tissue (cat GD450)+5 ul (100 ng) of MV5 contaminant digested        with DNAse I (Sigma)    -   6. Tissue (cat GD450)+5 ul (100 ng) of MV5 contaminant treated        with DNAZap (Ambion)    -   7. Tissue (cat GD450)+5 ul (100 ng) of MV5 contaminant washed        with water.

In seven 1.5 ml microfuge tubes, cat GD450 tissue and/or cat MV5microsatellite PCR product contaminant were mixed and left in contactfor 30 min at room temperature. In tubes 1, 2, and 3 no furthertreatment was performed. In tube 4, 10× Promega Buffer B (3 μl), 0.5 μlPromega Hae III restriction enzyme (9 units) and 21.5 μl of water wereadded and incubated at 37° C. for 1 hour. In tube 5, 10× Promega BufferC (3 μl), 0.5 μl DNAse I (˜50 units) and 21.5 μl water were added andincubated at 37° C. for 1 hour. In tube 6, DNAZap Solution 1 (10 μl) wasadded to the tube immediately followed by 10 μl of DNAZap Solution 2(Ambion Pty Ltd). After approximately 10 seconds the tissue sample wasthoroughly rinsed with deionised water prior to further use. In tube 7,the tissue sample was washed twice 1 ml of water, dried with tissuepaper and transferred into a new tube. The tissue was then washed againwith 2×1 ml of water.

In every tube, the integrity of the tissue samples was preservedfollowing the treatments.

Following the above treatments 300 μl of Masterpure Tissue and CellLysis Buffer+50 μg Proteinase K were added (Epicentre Technologies). Thesamples were incubated overnight at 65° C. in a hybridization oven withrotation. After this treatment, no tissue was left in the tubes. RNA wasremoved by addition of 5 μg of RNAse A and incubation at 37° C. for 30min. Tubes were cooled on ice and 150 μl of MPC Protein PrecipitationReagent were added (Epicentre Technologies). The precipitate waspelleted by centrifugation at 10,000 g for 10 minutes at 4° C.Supernatants were transferred into new tubes.

After addition of 500 μl of isopropanol and centrifugation for 10minutes, the DNA pellet was washed with 70% EtOH and resuspended in 40μl of water.

PCR Reactions

Samples from tubes 1-7 were used in individual PCR amplifications todetect microsatellite loci from cat GD450. Amplifications of individualmicrosatellite loci were performed in either 10 μl or 20 μl reactionscontaining 1 μl of template nucleic acid solution from each of tubes 1-7according to the procedure of Menotti-Raymond et al (1999). The 10 μlreactions were approximated to contain 2.5 ng of contaminant ({fraction(1/40)}×100 ng) whilst the 20 μl reactions contained approximately 10 ngof contaminant (4×2.5 ng).

A PCR amplification control was performed where 1 ul of contaminant (20ng) was reamplified as above.

Results

The results (FIG. 2) demonstrate the presence of the substantiallycorrect profiles for MV5 contaminant and GD450 controls (Contaminant andSample 1 lanes, respectively) as well as animal positive controls(animals A, B and C). Significant stutter peaks were present in the MV5contaminant controls equivalent to alleles 25, 23 and 9. Additionallythe MV5 contaminant control contained a band at a size equivalent to anallele at 22. The exact identity of this band is unknown but it ispossibly the result of heteroduplex formation during PCR. In the GD450positive control stutter peaks at allele equivalent 23, 19 and 17 arepresent. Smaller are bands in GD450 at allele equivalents 11 and 9 arethe result of spillage from adjacent lanes.

In samples containing mixtures of cat GD450 genomic DNA and MV5microsatellite PCR product contaminant (mixture samples) only bandsrepresentative of the MV5 contaminant were present. Since thecontaminant amplified in these samples was added to cat GD450 tissueprior to genomic DNA extraction, the results show that contaminatingmicrosatellite PCR products were efficiently extracted during theisolation of DNA from tissue of cat GD450. These PCR productcontaminants could be amplified during subsequent PCR implication forcat GD450 microsatellite loci. The results further show that when 10 ngof contaminating microsatellite was added to 4.5 mg of tissue thecontaminating microsatellite PCR products were able to entirely mask thegenuine cat GD450 profile (eg: mixture samples).

With the exception of DNAse (mixture+DNAse) the treatments trialled toremove the contaminating microsatellite PCR products failed to have anysignificant effect on the level of contaminant amplified duringsubsequent amplification for cat GD450 microsatellite loci. In thesample treated with DNAse I, however, the microsatellite contaminant wasefficiently removed since no detectable cat MV5 bands were presentfollowing PCR amplification for cat GD450 microsatellite loci(mixture+DNAse I, 1 μL and 5 μL samples). However, there was also anabsence of GD450 bands suggesting that the treatment used alsocompletely removed genomic DNA from the sample.

This result demonstrated that the removal of contaminatingmicrosatellite PCR products from tissue is possible. However, it is noteasily achieved by either physical or chemical methods that arefrequently used to remove contaminating DNA. It also showed that thecomplete removal of contaminating microsatellite PCR products requiresadditions/modifications to both reagents and protocols in DNA extractionmethods often used for DNA fingerprinting studies. DNAse I was effectiveat removing contaminating microsatellite PCR products but the methodused in this study is not suitable for inclusion in a DNA extractionkit.

Example 3 Contamination of Human Nucleic Acid Samples

To confirm the contamination issue also affected other PCR DNA templateand primer sets, particularly those involving human samples similar totypical forensic samples, experiments were conducted which used humanbuccal swabs.

Materials/Methods

In these experiments commercially available buccal swabs, typically usedfor parentage, pathogy and also forensic purposes were used to collectcheek cells from a human source. These buccal swabs were transported toa separate laboratory where they were impregnated with previouslyprepared mixture of PCR amplicons derived from DNA isolated from anunrelated human source. This mixture consisted of PCR amplicons derivedfrom 6 separate microsatellite loci (FIBRA, D8S1179, D5S818, D7S820,D13S317, D19S253).

The human PCR amplicons were diluted with water to a concentration of 10ng/μl and 1 μl of the undiluted as well as 10⁻³, 10⁻⁶, and 10⁻⁹dilutions were prepared and separately added to individual buccal swabs.Where appropriate these swabs were used to collect cheek and other cellsby wiping on the inside of the cheek as per standard operationalprocedures for collecting buccal samples prior to the addition of thecontaminating PCR amplicons. DNA was then extracted from the materialassociated with the swabs using a rapid DNA extraction procedure knownto work well with buccal swab samples for parentage analysis.

The purified DNA was then subjected to PCR amplification using primersspecific for the above loci (FIBRA, D8S1179, D5S818, D7S820, D13S317,D19S253) using standard well established conditions for theamplification of these loci.

Results

The results are depicted in FIG. 3. Samples (see Table hereunder) weresubjected to DNA extraction and FIBRA locus specific amplification asindicated. Lanes 1-4 were samples with PCR amplicon dilutions only. ThePCR amplicons contained only alleles 1 and 3 (Lane 11). Lanes 6-9 areamplification products from DNA extracts containing both cellular andcontaminating PCR amplicons. The cellular DNA had a profile consistingof alleles 2 and 4 (Lane 10). Lane 1 Swab containing undiluted PCRamplicons only. Lane 2 Swab containing 10⁻³ dilution of amplicons only.Lane 3 Swab containing 10⁻⁶ dilution of amplicons only. Lane 4 Swabcontaining 10⁻⁹ dilution of amplicons only. Lane 5 BLANK Lane 6 Swabcontaining cells and undiluted PCR amplicons. Lane 7 Swab containingcells and 10⁻³ dilution of amplicons. Lane 8 Swab containing cells and10⁻⁶ dilution of amplicons. Lane 9 Swab containing cells and 10⁻⁹dilution of amplicons. Lane 10 Swab containing cells only as positivecontrol (alleles 2 and 4). Lane 11 Re-amplified PCR amplicons only(alleles 1 and 3).

The results of the next procedure are depicted in the FIG. 4. Samples(see Table hereunder) were subjected to DNA extraction and a tri-plexamplification reaction with primers specific for the D5S818, D7S820, andD13S317 loci was performed as indicated. Lanes 1-4 were samples with PCRamplicon dilutions only. The PCR amplicons contained D7S820 allele 1,D13S317 alleles 1 and 2, D5S818 allele 2 (Lane 11). Lanes 6-9 containsamplification products from samples with both cellular DNA andcontaminating PCR amplicons. The cellular DNA had a profile consistingof D7S820 allele 2, D13S317 allele 3, D5S818 allele 1 (Lane 10). Lane 1Swab containing cells and undiluted PCR amplicons. Lane 2 Swabcontaining cells and 10⁻³ dilution of amplicons. Lane 3 Swab containingcells and 10⁻⁶ dilution of amplicons. Lane 4 Swab containing cells and10⁻⁹ dilution of amplicons. Lane 5 BLANK Lane 6 Swab containing cellsand undiluted PCR amplicons. Lane 7 Swab containing cells and 10⁻³dilution of amplicons. Lane 8 Swab containing cells and 10⁻⁶ dilution ofamplicons. Lane 9 Swab containing cells and 10⁻⁹ dilution of amplicons.Lane 10 Swab containing cells only as positive control. Lane 11Re-amplified PCR amplicons only.

The results of the next procedure are depicted in the FIG. 5. Samples(see Table hereunder) were subjected to DNA extraction and a bi-plexamplification reaction with primers specific for the D8S1179 and D19S253loci was performed as indicated. Lanes 1-4 were samples with PCRamplicon dilutions only. The PCR amplicons contained only D8SI 179alleles 1 and 2, D19S253 alleles 1 and 2 (Lane 11). Lanes 6-9 containsamplification products from samples with both cellular DNA andcontaminating PCR amplicons. The cellular DNA had a profile consistingof D8S1179 allele 3, D19S523 alleles 1 and 2 (Lane 10). Lane 1 Swabcontaining cells and undiluted PCR amplicons. Lane 2 Swab containingcells and 10⁻³ dilution of amplicons. Lane 3 Swab containing cells and10⁻⁶ dilution of amplicons. Lane 4 Swab containing cells and 10⁻⁹dilution of amplicons. Lane 5 BLANK Lane 6 Swab containing cells andundiluted PCR amplicons. Lane 7 Swab containing cells and 10⁻³ dilutionof amplicons. Lane 8 Swab containing cells and 10⁻⁶ dilution ofamplicons. Lane 9 Swab containing cells and 10⁻⁹ dilution of amplicons.Lane 10 Swab containing cells only as positive control. Lane 11Re-amplified PCR amplicons only.

The results demonstrate that the profiles of the cheek and other cellspresent on the buccal swabs differed from the contaminant PCR ampliconsfor all loci examined except D19S253 (FIG. 3—Lanes 10 and 11; FIG.4—Lanes 10 and 11; FIG. 5—Lanes 10 and 11). This enabled simpleidentification of genuine profiles derived from the cellular materialand the profile from any added PCR amplicons.

In order for amplification of the added PCR amplicons to occur they mustbe extracted with similar efficiency to the cellular DNA during DNAextraction procedures. The results show that PCR amplicons, when addedto a typical forensic sample such as a buccal swab, are extracted usingthe common DNA extraction protocol used here and are efficientlyamplified in subsequent PCR reactions for the appropriate loci. In allexperiments, regardless of the locus amplified, the contaminatingamplicons were able to completely mask the genuine profile of the cellsin the buccal swab (FIG. 3—Lanes 6 and 7; FIG. 4—Lanes 6 and 7; FIG.5—Lanes 6 and 7). This effect occurred with undiluted (10 ng/μl) and10⁻³ (10 pg/μl) samples (FIG. 3—Lanes 6 and 7; FIG. 4—Lanes 6 and 7;FIG. 5—Lanes 6 and 7).

Very little or no visible contamination with added PCR amplicons wasseen with 10⁻⁶ (10 fg/μl) and 10⁻⁹ (10 ag/μl) dilutions (FIG. 3—Lanes 8and 9; FIG. 4—Lanes 8 and 9; FIG. 5—Lanes 8 and 9) and even when nocellular DNA was present (FIG. 3—Lanes 1 to 4; FIG. 4—Lanes 1 to 4; FIG.5—Lanes 1 to 4). This indicates that the failure to amplify anycontaminating PCR amplicons present in the sample is due to theextremely low level (very few amplicon molecules) of the original sampleusing the PCR conditions used here. The use of a more sensitiveamplification and/or detection procedure may detect the low levels ofcontaminating PCR amplicons present in even these samples.

These results clearly demonstrate the potential for contaminating PCRamplicons to mask or confuse the DNA profile derived from a typicalsource of forensically important samples.

Example 4 Cleanup of Nucleic Acid Samples Using Micron CentriconUltracentrifugation

To demonstrate the principle of ultrafiltration as an example of aphysical means of removing contamination PCR amplicons from genomic DNA(or sample) Amicon YM-100, Microcon-100 microconcentrators (nominalmolecular weight cut-off of 100 kDa, corresponding to 300 nt ofsingle-stranded DNA or 125 bp of double-stranded DNA: MilliporeApplication note AN023EN00), were used to separate PCR amplicons fromcellular genomic DNA.

Many small PCR amplicons such as oligonucleotides used for profilingwith single nucleotide polymorphisms will pass directly through thesemicroconcentrators. Other PCR amplicons such as those from typicalforensic analyses including the commercially available DNA profilingkits (as well as many other microsatellite and mitochondrial DNAprofiling analyses) are all larger than 125 bp. Consequently, thismethod will not separate genomic DNA templates from double strandedthese larger amplified DNA products.

However, as the nominal molecular weight cut-off for single stranded DNAis approximately twice the double stranded DNA cut-off (YM-100; 300 and125, respectively) denaturation of the template will allow many, andperhaps all (depending on the size of PCR amplicons) to pass through themicroconcentrator, allowing separation of the contaminating PCRamplicons and high yield recovery of the important sample genomic DNA.

Methods

DNA was isolated from 2 cattle blood samples and subjected to PCRamplification using BM2113 microsatellite primers with the sequence;5BM2113 forward - (5′) GCT GCC TTC TAC CAA ATA CCC (3′) BM2113 reverse -(5′) CTT CCT GAG AGA AGC AAC ACC (3′).

The amplification reaction consisted of the following (per reaction),BM2113 forward primer:   1 μl BM2113 reverse primer:   1 μl 10× DNApolymerase buffer (Promega): 2.5 μl dNTPs (200□M each):   4 μl Magnesiumchloride (25 mM): 2.5 μl Taq polymerase (Applied Biosystems):   1 μldistilled water:  11 μl Template DNA (approximately 100 ng):   2 μlTOTAL  25 μl

The reaction mix was then subjected to the following thermal cycle in anApplied Biosystems GeneAmp PCR System 9700 thermal cycler to amplify thetemplate DNA present.

Denaturation: 95° C./15 minutes

Annealing and extension (31 cycles):

94° C./45 seconds (100% ramp rate)

61° C./45 seconds (50% ramp rate)

72° C./60 seconds (80% ramp rate)

Final extension: 72° C./60 minutes

Final Step: 25° C./2 hours

Hold: 4° C. until required.

The resultant amplification products were then separated by highresolution agarose gel electrophoresis (Fisher Biotec Ultra HighResolution Agarose) in 6% gels until the required separation wasachieved.

The cattle DNA samples used were amplified using the BM2113 primers todetermine the alleles present in each animal (FIG. 6—Lanes 2 and 3).This showed the animals had easily differentiated loci. The PCR product(1 μl of {fraction (1/10,000)} dilution of the PCR reaction,approximately 0.1 pg) from animal 1 (FIG. 6—Lane 2) were then added toapproximately 10 ng of template genomic DNA from animal 2. The mixtureof genomic DNA and PCR amplicons was then subjected to amplification aspreviously using BM22113 primers. Analysis of the resultant products(FIG. 6—Lane 4) demonstrated the presence of alleles from both animal 1(allele 1) and animal 2 (allele 2).

To separate contaminating PCR amplicons and cellular genomic DNA byultrafiltration, a Microcon-100 sample reservoir was placed into amicrocentrifuge tube and the reservoir filled with 400 μl TE(10 mMTris-HCl pH8.0, 0.1 mM EDTA) and up to 50 μl of untreated or treatedamplicon/genomic DNA mixture. The treatments consisted of one of either,heat denaturation at 95° C. for 1-15 minutes in deionised water orformamide solution (10 mM NaOH, 95% deionised formamide), or alkalinedenaturation with sodium hydroxide (0.2M) for 5 minutes followed byneutralisation.

After centrifugation at 500 g for 15 min in an Eppendorf microcentrifuge(Model 5415C) at room temperature, another 400 μl of TE was added to thesample reservoir and centrifugation continued for approximately 15minutes or until the volume in the retentate cup was reduced to about 20μl. To recover retained DNA larger than the nominal molecular weightcut-off, the reservoir was removed and inverted into a newmicrocentrifuge tube then centrifuged at 500 g for 2 min. The resultingDNA (approximately 20 μl) was used in subsequent PCR amplificationstudies with BM2113 to identify the source (contaminating amplicon orcellular DNA) of the DNA present in the concentrate solution.

Results

Without treatment to render the DNA single stranded, the PCR ampliconsand cellular genomic DNA were efficiently retained in the concentrate(FIG. 6—Lanes 5 and 6). This is expected as the as the microconcetratorsare designed to retain genomic DNA and the added PCR amplicons areapproximately 140 bp in size whilst the cut-off of the microconcentratormembrane is approximately 125 bp.

However, treatment of the DNA mixture prior to PCR by heating at 95° C.for 10 minutes to render the DNA single stranded, followed by immediateapplication to the microconcentrator and centrifugation as above,resulted in almost complete removal of the added amplicon DNA (FIG.6—Lanes 7 and 8). In FIG. 6 only the alleles representative of cellulargenomic DNA are present (Lanes 7 and 8).

This clearly demonstrates that contaminating PCR amplicons can beremoved by ultrafiltration using a microconcentrator with an appropriatenominal molecular weight cut-off. For amplicons with sizes that are notsuited to the Amicon YM-100 Microcon-100 microconcentrators othermicroconcentrators with a suitable cut-off can be used.

Example 5 Cleanup of Nucleic Acid Samples Using Sodium Bisulfite

To demonstrate the principle of chemical modification of the DNA as anexample of a chemical means of removing contamination PCR amplicons fromgenomic DNA (or sample) DNA was treated with sodium bisulfite. Thisreagent is commonly used to convert cytosine, but not 5-methylcytosine,in DNA into uracil. It is particularly useful for DNA methylationstudies (Granau et al, Nucleic Acids Research (2001) Vol. 29, No. 13;Raizis et al, Analytical Biochemistry (1995) Vol. 226, 161-166.) Thisfeature also makes it a useful reagent for the specific removal of PCRamplicons from genomic DNA as the PCR amplicons contain only cytosine(there is no methylation of cytosines during PCR) whilst the humancellular genomic DNA (and DNA from many other organisms) will contain aproportion of methylated cytosines which are susceptible to conversionto uracil.

Because of the specific interaction between cytosine and sodiumbisulfite the conversion of cytosine to uracil under the conditions usedproceeds very rapidly with single stranded DNA, but only slowly when theDNA is double stranded. Since PCR amplicons are relatively smallcompared to genomic DNA they are more readily denatured into singlestranded form. This will facilitate the cytosine to uracil conversion inany PCR amplicons present whilst rendering the genomic DNA considerablymore resistant to such conversion.

As demonstrated in laboratory contamination control of PCR ampliconcontamination using uracil glycosylase, this enzyme is able to removethe uracil from DNA rendering the DNA incapable of amplification insubsequent PCR reactions.

By treating the converted DNA from the sodium bisulfite reaction withuracil glycosylase the contaminating PCR amplicons in a sample shouldsimilarly become resistant to subsequent amplification by PCR.

Methods

The methods and reagents used for the bisulfite treatment of DNA wereessentially as described by Raizis et al (Analytical Biochemistry (1995)Vol. 226, 161-166). A 5M sodium bisulfite solution was prepared byadding 1.9 g of sodium metabisulfite to 2.5 ml of distilled water andvortexing for 1 minute. To this solution 0.7 ml of 0.2M sodium hydroxideand 0.5 ml of hydroquinone were added. The solution was vortexed untilcompletely dissolved. The pH was adjusted to 5.0 with sodium hydroxideand the final volume was made up to 4 ml with distilled water.

To obtain single-stranded DNA, the DNA (approx 0.5 μg) was incubated in0.3 M NaOH at 37° C. for 20 min. These conditions were chosen as initialstudies demonstrated that PCR amplicons appeared to be preferentiallydenatured using these conditions. The reaction volume was adjusted to 30μl with sterile water and 3 μl of 2 M sodium hydroxide was added. TheDNA was incubated at 45° C. for 15 minutes and following this incubationfreshly prepared bisulfite solution was added (200 μl/μg DNA) directlyto the denatured DNA. The mixture was incubated at 50° C. for 4 hours.

The DNA was neutralised and precipitated by the addition of 0.5 volumesof 3M sodium acetate (pH7.0), 2.3 volumes of distilled water, and anequal volume of isopropanol. The mixture was incubated on ice for atleast 30 minutes. The DNA was then pelleted by centrifugation in amicrocentrifuge for 15 minutes.

The supernatant was discarded and the pellet was resuspended in 300 μldistilled water, 30 μl 3M sodium acetate (pH7.0) and reprecipitated with2 volumes of ethanol. The DNA was then pelleted by centrifugation in amicrocentrifuge for 15 minutes. The pellet was redissolved in 200 μl ofsodium hydroxide (0.2M) and incubated at room temperature for 15minutes.

The DNA was then precipitated by the addition of 0.5 volumes of 7.5Mammonium acetate and 2 volumes of ethanol. Following microcentrifugationfor 30 minutes the supernatant was discarded and the DNA pellet wasredissolved in 50 μl of distilled water. The DNA was then analysed byagarose gel electrophoresis or subjected to PCR amplification.

To demonstrate this method DNA was isolated from 2 cattle blood samplesand subjected to PCR amplification using SPS 115 microsatellite primers.These have the sequence; SPS 115 forward - (5′) AAA GTG ACA CAA CAG CTTCTC CAG (3′) SPS 115 reverse - (5′) AAC GAG TGT CCT AGT TTG GCT TGT(3′).

The amplification reaction consisted of the following (per reaction),SPS 115 forward primer:   1 μl SPS 115 reverse primer:   1 μl 10× DNApolymerase buffer (Promega): 2.5 μl dNTPs (200□M each):   4 μl Magnesiumchloride (25 mM): 2.5 μl Taq polymerase (Applied Biosystems):   1 μldistilled water:  11 μl Template DNA (approximately 100 ng)   2 μl TOTAL 25 μl

The reaction mix was then subjected to the following thermal cycle in anApplied Biosystems GeneAmp PCR System 9700 thermal cycler to amplify thetemplate DNA present.

Where required Uracil-DNA-glycosylase (1 μl—Fisher Biotec) was added tothe PCR reaction mix prior to the initial denaturation step andincubated at 37° C. for 15-60 minutes to digest any uracil containingDNA.

Denaturation: 95° C./15 minutes

Annealing and extension (31 cycles):

94° C./45 seconds (100% ramp rate)

61° C./45 seconds (50% ramp rate)

72° C./60 seconds (80% ramp rate)

Final extension: 72° C./60 minutes

Final Step: 25° C./2 hours

Hold: 4° C. until required.

The resultant amplification products were then separated by highresolution agarose gel electrophoresis (Fisher Biotec Ultra HighResolution Agarose) in 6% gels until the required separation wasachieved.

Results

DNA isolated from animal 1 and animal 2, respectively was subjected toPCR amplification with SPS 115 primers. Animal 1 (FIG. 7—Lane 2) hadonly allele 2 whilst animal 2 had allele 1 and allele 2 (FIG. 7—Lane 3).These were readily differentiated using 6% high resolution agarose gelelectrophoresis allowing discrimination of the source of the alleles(animal 1 or animal 2).

The PCR product (1 μl of {fraction (1/10,000)} dilution of the PCRreaction, approximately 0.1 pg) from animal 2 (FIG. 7—Lane 3) were thenadded to approximately 100 ng of template genomic DNA from animal 1. Themixture of genomic DNA and PCR amplicons was then subjected toamplification as previously using SPS 115 primers. Analysis of theresultant products (FIG. 7—Lane 4) demonstrated the presence of allelesfrom both animal 1 (allele 1) and animal 2 (allele 2).

To separate contaminating PCR amplicons and cellular genomic DNA, theamplicon/genomic DNA mixture above treated with bisulfite anduracil-DNA-glycosylase according to the methods above. In these studiesthe PCR amplicons were diluted {fraction (1/1000)} and 1 μl was added toapproximately 1 μg of template genomic DNA. This was to compensate forpotential losses of DNA during the processing with bisulfite.

The DNA mix was treated with bisulfite with (FIG. 7—Lanes 7 and 8) orwithout (FIG. 7—Lanes 5 and 6) initial template DNA denaturation. Insamples that were not denatured both alleles 1 and 2 were presentfollowing amplification/digestion (FIG. 7—Lanes 5 and 6). There appearedto be little loss of allele 1 associated exclusively with animal 2. Asallele 1 was present in the sample only through the addition ofcontaminating PCR amplicons derived from animal 2 this demonstrates thatthere had been little detectable loss of the contaminating PCR ampliconsfrom the mixture.

In contrast where the sample template DNA had been denatured prior tosodium bisulfite treatment, following PCR amplification/digestion, therewas significant loss of allele 1 (FIG. 7—Lanes 7 and 8) although allele2 was still present at detectable levels. This suggests that thetreatment had resulted in the preferential removal of contaminating PCRamplicons from the mixed sample.

The results show that it is possible to use sodium bisulfite incombination with uracil-DNA-glycosylase to effectively removecontaminating PCR amplicons.

The present invention includes modifications and adaptations apparent tothose skilled in the art.

Throughout the specification, unless the context requires otherwise, theword “comprise” or variations such as “comprises” or “comprising”, willbe understood to imply the inclusion of a stated integer or group ofintegers but not the exclusion of any other integer or group ofintegers.

1. A method of analysing a nucleic acid sample obtained from a sitecomprising the step of pretreating the sample to remove or inactivatecontaminating nucleic acids originating from the site.
 2. A methodaccording to claim 1 wherein the contaminating nucleic acid isdeoxyribonucleic acid (DNA), ribonucleic acid (RNA), locked nucleic acid(LNA) or protein nucleic acid (PNA).
 3. A method according to claim 1wherein the contaminating nucleic acid is particularly well adapted foramplification via PCR or some other amplification process.
 4. A methodaccording to claim 3 wherein the contaminating nucleic acid is anamplicon derived from a PCR or another DNA amplification process.
 5. Amethod according to claim 1 wherein the contaminating nucleic acid isdegradation resistant.
 6. A method according to claim 1 wherein thecontaminating nucleic acid is synthetic.
 7. A method according to claim1 wherein the pre-treatment comprises treating the sample topreferentially remove or inactivate nucleic acids that are free orsubstantially free from other cell components.
 8. A method according toclaim 7 wherein the pre-treatment is one or more treatments selectedfrom the group comprising: (i) enzymic treatments; (ii) physicaltreatments; and (iii) chemical treatments.
 9. A method according toclaim 8 wherein the enzymic treatments comprise contacting the samplewith DNAses, RNAses, exonucleases and/or 25 endonucleases.
 10. A methodaccording to claim 8 wherein the physical treatments comprisecentrifugation, washing, filtration and/or chromatography such as gelfiltration chromatography.
 11. A method according to claim 8 wherein thechemical treatments comprise the use of sodium hydroxide, sodiumhypochlorite, sodium metabisulphite or ammonium metabisulphite,detergents and/or proprietary products designed to remove nucleic acidsform surfaces.
 12. A method according to claim 1 wherein the method ofanalysing the nucleic acid sample is PCR, mitochondrial DNA sequencing,single nucleotide polymorphism (SNP) analysis and low copy number PCR.13. A method according to claim 1 wherein the pre-treatment comprisesremoving cell bound contaminating nucleic acids from the sample.
 14. Amethod according to claim 13 wherein the cell bound contaminatingnucleic acid is particularly well adapted for amplification via PCR orsome other amplification process.
 15. A method according to claim 13wherein the contaminating nucleic acid is of bacterial origin.
 16. Amethod according to claim 15 wherein the contaminating nucleic acid isbacteria engineered to contain at least one multicopy plasmid comprisingat least one amplicon.
 17. A method according to claim 13 wherein thecell bound contaminating nucleic acid is removed by exposing the nucleicacid in the cells and then removing the nucleic acid.
 18. A methodaccording to claim 17 wherein the nucleic acid is exposed by lysing thecells.
 19. A method according to claim 17 wherein the nucleic acid isremoved using the pre-treatment steps of claim
 7. 20. A nucleic acidanalysis kit comprising a means to remove a nucleic acid contaminantfrom a sample to be subjected to analysis.
 21. A kit according to claim20 wherein said means comprises a labelled probe adapted to bind to thecontaminant and thus aid in its removal.
 22. A kit according to claim 20wherein said means comprises an enzyme or chemical that can be added tothe sample and inactivate of remove the contaminant preferentially orselectively relative to a target nucleic acid. 23-42. (canceled)